Recombinant heterodimeric human fertility hormones, and methods, cells, vectors and DNA for the production thereof

ABSTRACT

Biologically active heterodimeric human fertility hormones composed of two different subunits, each subunit being synthesized in the same cell transformed by at least one cell expression vector having heterologous DNA encoding each subunit with each subunit being controlled by a separate promoter. Preferred human fertility hormones include hCG, hLH and hFSH.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.07/515,481, filed Apr. 27, 1990, now abandoned, which application is acontinuation-in-part of both Ser. No. 07/323,772, filed Mar. 15, 1989,now abandoned, and Ser. No. 06/696,647, filed Jan. 30, 1985, now U.S.Pat. No. 4,923,805, the entire contents of both of which are herebyincorporated herein by reference. Ser. No. 07/323,772 is a continuationof Ser. No. 06/548,228, filed Nov. 2, 1983 now U.S. Pat. No. 4,840,896.Ser. No. 06/696,647 is a continuation-in-part of said Ser. No.06/548,228.

BACKGROUND OF THE INVENTION

The present invention relates to the use of recombinant DNA techniquesto produce heterodimeric human fertility hormones.

Various polypeptide chains have been expressed, via recombinant DNAtechnology, in host cells such as bacteria, yeast, and culturedmammalian cells. Fiddes, J. C. and Goodman, H. M. Nature, vol. 281, pp.351-356 (1979) and Fiddes, J. C. and Goodman, H. M., Nature, vol. 286,pp. 684-687 (1980) describe the cloning of, respectively, the alpha andbeta subunits of human choriogonadotropin (hCG).

Sugimoto U.S. Pat. Nos. 4,383,034, 4,383,035 and 4,383,036 describeprocesses for producing FSH, LH and hCG, respectively, in which humanlymphoblastoid cells are implanted into a laboratory animal, harvestedfrom the animal, and cultured in vitro; accumulated hormone is thenharvested from the culture. This technique is not capable of producingsubstantially pure hormone free of any other human fertility hormone.

Cohen et al, U.S. Pat. No. 4,468,464 mentions the production offertility hormones by recombinant DNA techniques. However, Cohen et alonly uses a prokaryotic system which cannot produce biologically activehuman fertility hormone.

Pierce et al, Ann. Rev. Biochem., 50, 465-95 (1981) states that thealpha and beta subunits of LH are known to associate in vitro. Thesubunits referred to in this paper are obtained by dissociatingnaturally occurring dimeric hormone and allowing the units toreassociate. Such a disclosure does not permit the prediction that whensynthesized by non-specialized cells transformed with recombinant DNA,the subunits would be properly glycosylated and folded for associationso as to produce a biologically active hormone.

While many human proteins have been produced by recombinant DNAtechniques the production of biologically active heterodimeric hormonesby such techniques has not heretofore been accomplished. Heterodimericfertility hormones are produced in the human body by highly specialized,differentiated cells which have evolved over a long period of time tocarry out the specialized function of producing each particular hormone.The mechanism by which post-translational heterodimeric assembly occursintracellularly in these differentiated cells is not known, but it isknown that proper assembly is necessary for biological activity.Undifferentiated cells do not, as far as is known, normally producehormones. Thus, whether or not a biologically active heterodimerichormone could be produced in undifferentiated cells transformed with DNAencoding the alpha and beta subunits was totally unpredictable.

SUMMARY OF THE INVENTION

The present invention stems from the unpredictable discovery thatbiologically active heterodimeric human fertility hormones can beproduced in eukaryotic cells transformed by vectors containing the alphaand beta subunits of the hormone controlled by separate promoters. Whilealpha and beta subunits produced in separate cultures will notreassociate to form biologically active hormones, it has unexpectedlybeen discovered that when both subunits are produced in the same cell, ahormone is expressed which is biologically active.

Thus, the present invention includes the substantially pureheterodimeric human fertility hormones which can now be made totallyfree of other fertility hormones as well as any other human proteins. Italso includes the process for production of such hormones, eukaryoticcells which have been transformed to so produce the hormones and vectorscontaining the DNA of both the alpha and beta subunits.

The present invention also includes the DNA encoding the beta subunitsof hLH and hFSH, including cDNA coding for the beta subunit of humanFSH, expression vectors containing such DNA and cells transfectedtherewith. The invention also includes DNA derivatives according to thegenetic code which on expression code for the beta subunit of human FSHaccording to the present invention. The polypeptide structure of thebeta subunit of hFSH has never before been accurately set forth.

Thus, in general, the present invention features, in one aspect, asubstantially pure heterodimeric human fertility hormone composed of twodifferent subunits and the process for the production thereof by meansof which the two subunits are synthesized by single cell line, each cellhaving been transformed by an expression vector containing heterologousDNA encoding both subunits under control of separate promoters or twoexpression vectors each containing heterologous DNA encoding theseparate subunits. The cell line is composed of eukaryotic cells whichpermit appropriate post-translational modification of the subunits suchthat the formed protein is biologically active. Because of therecombinant DNA technique which is used, the hormones produced aresubstantially pure and free of any other fertility hormones or any otherhuman proteins. The preferred fertility hormones which are produced inaccordance with the present invention are hCG, luteinizing hormone (LH)and follicle stimulating hormone (FSH).

In another aspect, the present invention features a cell transformed byat least one expression vector, which cell is capable of producing abiologically active heterodimeric protein that is encoded at least inpart by the vector. In preferred embodiments: a second expression vectorencodes a second portion of the protein or at least two subunits of theprotein are encoded by a single expression vector; the vectors areautonomously replicating, preferably a replicating virus or a plasmid;the cell is a mammalian cell, such as a monkey or mouse cell;transcription of the different subunits is under the control of the SV40late promoter; transcription of the alpha subunit of the protein isunder the control of the SV40 early promoter and transcription of thebeta subunit is under control of the mouse metallothionein promoter, ortranscription of both subunits is under the control of the mousemetallothionein promoter; and the expression vector which includes themouse metallothionein promoter also includes at least the 69%transforming region of the bovine papilloma virus (BPV) genome.

In another aspect, the invention features an autonomously replicatingexpression vector including two genes encoding two differentheterologous proteins, the genes being under the control of twodifferent promoters, most preferably a metallothionein promoter and aBPV promoter; the use of different promoters advantageously minimizesthe possibility of deleterious recombinations.

In a further aspect, the invention features the DNA for the betasubunits of hLH and hFSH.

As used herein, "subunit" refers to a portion of a protein, whichportion, or homologue or analogue thereof, is encoded in nature by adistinct mRNA. Thus, for example, a heavy chain and a light chain of anIgG immunoglobulin are each considered a subunit. Insulin, on the otherhand, is composed of two chains which are not considered subunits,because both are, in nature, encoded by a single mRNA, and cleavage intotwo chains naturally occurs only after translation.

The term "expression vector" refers to a cloning vector which includesheterologous (to the vector) DNA under the control of control sequenceswhich permit expression in a host cell. Such vectors include replicatingviruses, plasmids, and phages. Preferred vectors are those containing atleast the 69% transforming region, and most preferably all, of thebovine papilloma virus genome.

The invention permits the production of a biologically activeheterodimeric human fertility hormone from a single culture oftransformed cells which hormone undergoes, in the culture,post-translational modification, e.g. glycosylation and proteolyticprocessing, for biological activity and stability.

In preferred embodiments, each expression vector is autonomouslyreplicating, i.e., not integrated into the chromosome of the host cell.The use of autonomously replicating expression vectors preventsundesirable influence of the desired coding regions by control sequencesin the host chromosome.

Other advantages and features of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

We turn now to the preferred embodiments of the invention, first brieflydescribing the drawings thereof.

FIG. 1 is a diagrammatic illustration of the construction of the plasmidp alpha SVHVPl, which contains the alpha hCG cDNA clone, portions ofSV40 viral DNA, and sequences of the plasmid pBR322.

FIG. 2 is a diagrammatic illustration of the construction of plasmid pbeta SVVPl, which incorporates the beta hCG cDNA clone, regions of SV40DNA and a portion of pBR322 including the region conferring resistanceto ampicillin on host E. coli.

FIG. 3 is a diagrammatic illustration of the construction of the plasmidp alpha beta SVVPl in which the alpha and beta hCG cDNA clones areinserted into SV40 DNA.

FIG. 4 is a diagrammatic illustration of the construction of theplasmids pRF375 and pRF398.

FIG. 5 is a diagrammatic illustration of the construction of the plasmidRF398 alpha t₂.

FIG. 6 is a diagram illustrating the location of an 88 bp probe withinthe beta hCG cDNA clone.

FIG. 7 illustrates the beta LH restriction map, and the pieces used inthe construction shown in FIG. 8.

FIG. 8 is a diagrammatic illustration of the construction of a plasmid,LH520H/B, containing the complete mature beta LH cDNA clone.

FIG. 9 is a diagrammatic illustration of the construction of the viralvector p alpha LHSVVPl.

FIG. 10 is a diagrammatic illustration of the construction of theBPV-containing plasmid pCL28XhoLHBPV, encoding the beta subunit of LH.

FIG. 11 is a partial restriction map of the lambda clone 15B and thebeta FSH-containing 6.8 kb EcoRI-BamHI fragment that is inserted intopBR322.

FIG. 12 is a partial restriction map of the beta FSH coding region andthe BamHI fragment that is inserted into a BPV based expression vector.

FIG. 13 is a diagrammatic illustration of the construction of theBPV-containing plasmid CL28FSH2.8BPV, encoding the beta subunit of FSH.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The cloning vectors of the invention have the general structure recitedin the Summary of the Invention, above. Preferred vectors have thestructures shown in the Figures, and are described in more detail below.

Construction of Cloning Vectors Isolation of cDNA Clones Encoding theAlpha and Beta Subunits of hCG

All of the techniques used herein are described in detail in Maniatis etal, (1982) Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory), hereby incorporated by reference.

RNA is extracted from placental tissue by the following method.Homogenization of the tissue is carried out in a 1:1 mixture ofphenol:100 mM Na-acetate (pH 5.5) containing 1 mM EDTA, that has beenwarmed to 60° C. for 20 min. After cooling on ice for 10 min., thephases are separated by centrifugation. The hot phenol extraction isrepeated twice more followed by two extractions with chloroform.

RNA is precipitated from the final aqueous phase by the addition of 2.5volumes of ethanol.

In order to enrich for poly A⁺ mRNA, placental RNA is passed over oligo(dT)-cellulose in 0.5M NaCl buffered with 10 mM Tris-HCl, pH 7.5, andwashed with the same solution. Poly A⁺ mRNA is eluted with 10 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.05% SDS and precipitated twice withethanol. Typical initial yields are 1.5-2.0 mg of total RNA per g oftissue, of which about 2% is poly A⁺ mRNA.

Placental cDNA libraries are constructed by reverse transcription ofplacental mRNA, second strand synthesis using E. coli DNA polymerase I(large fragment), treatment with S1 nuclease, and homopolymer tailing(dC) with terminal deoxynucleotidyl transferase; all such procedures areby conventional techniques.

In a typical preparation, 20-30% conversion of mRNA to single strand(ss) cDNA; 70% resistance to digestion with nuclease S1 after secondstrand synthesis; and dC "tails" of ten to twenty-five bases in length,are obtained. These cDNA molecules are then annealed to DNA fragments ofthe plasmid pBR322, which has been digested with PstI, and to which dG"tails" have been added. These recombinant plasmids are then used totransform E. coli cells to generate a cDNA library (transformed cellsare selected on the basis of tetracycline resistance).

In order to identify the human alpha hCG clone, a 219 bp fragment of amouse alpha thyroid stimulating hormone (TSH) clone is used as ahybridization probe. This probe has 77% sequence homology with the humanclone. It is radioactively labeled by nick translation and hybridized tothe cDNA library under conditions that take into account the extent ofhomology. Strongly hybridizing clones are analysed by restrictionmapping and clones containing the complete coding sequence of alpha hCGare verified by DNA sequencing.

Construction of Plasmid p alpha SVHVPl

Referring to FIG. 1, in order to construct the plasmid alpha 970 H/B, acDNA clone containing the alpha hCG fragment is digested with NcoI. TheNcoI site, just 5' to the ATG codon signalling initiation oftranslation, is filled in and ligated to a synthetic HindIII linker.Similarly, the natural HindIII site in the 3' untranslated region of theclone is cut, filled in with E. coli DNA polymerase Klenow, and thenligated to a synthetic BamHI linker. This fragment is cloned into theplasmid pBR322 between its HindIII and BamHI sites to generate theplasmid alpha 574 H/B. This plasmid is digested with BamHI, treated withalkaline phosphatase, and ligated to the 396 bp Sau3A fragment of SV40DNA (from 0.07 to 0.14 map units) which has been isolated from apolyacrylamide gel. The ligation mix is used to transform E. coli toampicillin resistance and the desired plasmid, alpha 970 H/B, isidentified among the transformants.

The plasmid Q₂ 7 is constructed by cutting SV40 at its HsaII site,making flush ends by digestion with nuclease S1, ligating on EcoRIlinkers, digesting with EcoRI, and cloning the resulting 1436 bpfragment into the EcoRI site of pBR322.

Referring to FIG. 1, Q₂ 7 is digested completely with EcoRI andpartially with HindIII; the fragment from 0.72 to 0.94 map units isisolated and cloned into alpha 970 H/B, which has been digested withScoRI and HindIII and treated with alkaline phosphatase. The ligationmix is used to transform E. coli, and the desired plasmid, p alpha SVL,is identified among the transformants by restriction mapping.

p alpha SVL is digested with EcoRI and the fragment of SV40, with EcoRIends, extending from 0 to 0.72 map units, and containing the SV40 originof replication and the intact early region, is ligated to it to generatethe plasmid p alpha SVHVPl, which is isolated from E. colitransformants.

Construction of Plasmid p beta SVVPl

A 579 bp cDNA clone coding for beta hCG was obtained from John C. Fiddesat Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (Fiddeset al, Nature, vol. 286, pp. 684-687 (1980)). This fragment is ligatedat each end to synthetic BamHI linkers. After digestion by HgaIrestriction enzyme, the ends are filled in with Klenow DNA polymeraseand synthetic EcoRI linkers are ligated on so that an EcoRI site isabout 10 bp 5' to the ATG codon of the signal peptide coding sequence. ABamHI site is about 60 bp 3' to the nonsense codon marking the end ofthe coding sequence. Referring to FIG. 2, this 556 bp EcoRI-BamHIfragment is isolated and cloned into pBR322, between the EcoRI and BamHIsites, to give the plasmid p beta 556 R/B.

In order to construct the plasmid pSVHR (FIG. 2), SV40 DNA is partiallydigested with HindIII to yield linear molecules, digested with nucleaseS1 to make flush ends, ligated to synthetic EcoRI linkers and digestedwith EcoRI and BamHI. The fragment from 0.94 to 0.14 map units,containing the SV40 origin of replication and early region, is clonedinto pBR322 as an EcoRI-BamHI piece.

Referring still to FIG. 2, the EcoRI site of the plasmid p beta 556 R/Bis methylated in a reaction catalyzed by EcoRI methylase, followingwhich the plasmid is cut with NdeI. EcoRI linkers are ligated to the S1treated NdeI flush ends and activated by digestion with EcoRI, which isfollowed by digestion with BamHI.

The SV40 fragment of pSVHR from the EcoRI site to the BamHI site isisolated and ligated in a reaction mix containing the digestionfragments of p beta 556 R/B. Following ligation, the mix is digestedwith SalI to eliminate plasmids which have re-inserted the EcoRI (NdeI)to BamHI piece of pBR322. E. coli is transformed with the digestedligation mix and p beta SVVPl is identified and isolated.

Construction of the Plasmid p Alpha Beta SVVPl

Referring to FIG. 3, pBR322/Kpn is derived from pBR322 by inserting aKpnI linker into its unique EcoRI site, after this site is deleted bydigestion with EcoRI, followed by digestion with S1 nuclease.

Referring still to FIG. 3, SV40 DNA is digested with AvaII. Thestaggered ends of the resulting fragments are filled in by Klenow DNApolymerase to form flush ends, and the mixture is then fractionated on apolyacrylamide gel. The 682 base pair fragment (0.64 to 0.77 map units)containing the origin of replication and to the unique KpnI site isisolated from the gel, ligated to synthetic HindIII linkers, anddigested with HindIII and KsnI.

The resulting fragments are ligated to pBR322/Kpn. p266, which containsthe 266 base pair KpnI HindIII fragment, including the SV40 latepromoter region, is isolated. p266 is cut with HindIII and BamHI, andtreated with bacterial alkaline phosphatase.

Still referring to FIG. 3, p beta SVVPI/B is constructed as follows: pbeta SVVPI (FIG. 2) is cut with EcoRI, followed by ligation to eliminatepBR322 sequences. Subsequently, this DNA is cut with BamHI and clonedinto the BamHI site of pBR322.

The resulting plasmid, p beta SVVPI/B, is then digested with HindIII andBamHI and the 1003 base pair HindIII-BamHI fragment is ligated into p266to yield the plasmid p beta VPl 266, in which the beta hCG cDNA ispositioned downstream from the SV40 late promoter in such a way that itsRNA transcript would be spliced as if it were the viral VP1 transcript.

The alpha hCG cDNA is inserted into p beta VPl 266 as a HindIIIfragment, which has been cut at its HindIII site and treated withbacterial alkaline phosphatase. E. coli transformants derived from thisligation are screened by restriction mapping, and plasmids are isolatedthat have the desired structure, in which the alpha hCG cDNA hasreplaced VP2 in the correct orientation, followed downstream by the betahCG cDNA, which has replaced VP1.

One such isolated plasmid, p alpha beta VP1, is used to complete theconstruction of p alpha beta SVVPl. The plasmid is cut with KpnI, andthe full SV40 genome, cut with KpnI, is inserted by ligation into thissite. Following transformation of E. coli, a plasmid with the requiredstructure, p alpha beta SVVPl, is isolated. This plasmid contains DNAencoding both the alpha and beta subunits of hCG, and thus is capable ofdirecting the expression, in host mammalian cells, of both subunits,whereby biologically functional, glycosylated heterodimeric hCG isproduced (glycosylation occurs post-translationally).

Construction of Plasmids pRF 375 and pRF 398

Referring to FIG. 4, the plasmid CL28 (identical to plasmid JYMMT(E);Haner et al., J. Mol. Applied Gen., 1, 273-288 (1983)), containing themurine metallothionein promoter, SV40 DNA, and pBR322 sequences, is cutwith the restriction endonuclease Bgl II. At this site are inserted cDNAclones of either alpha hCG or beta hCG, containing untranslated regionsof about 10 and 30 bp at their 5' and of about 220 and 60 bp at their 3'ends. These clones have been genetically engineered by the addition ofsynthetic BamHI linkers at their termini.

The resulting plasmids pRF 302 (alpha) or pRF 394 (beta) are digestedwith restriction enzymes BamHI and SalI to release the SV40 DNAsequences.

Plasmid pB2-2, which contains the entire BPV genome, and some pBR322sequences, is digested with BamHI and SalI to yield the BPV genome withBamHI/SalI ends; this fragment is ligated into pRF 302 (alpha) and pRF394 (beta) containing the metallothionein-hCG sequences.

Following transformation of E. coli, plasmids pRF 375 and pRF 398 areidentified and isolated. They encode alpha hCG or beta hCG,respectively, under the control of the mouse metallothionein promoter.

Construction of the Plasmid RF 398 alpha t₂

Referring to FIG. 5, the plasmid p alpha t₂ is derived by cloning thealpha hCG 574 HindIII fragment into plasmid pVBt2 (V. B. Reddy et al.,PNAS, 79, 2064-2067, 1982). p alpha t₂, which contains the alpha hCGcDNA under the control of the SV40 early promoter, is digested withEcoRI. The 5' overhangs are removed by S1 nuclease digestion prior tothe addition of synthetic BamHI linkers by blunt end ligation.

Plasmid RF 398 (FIG. 4) is digested with BamHI and treated withbacterial alkaline phosphatase. The 1735 base pair BamHI fragment of palpha t₂ is inserted in to RF 398. The resulting plasmid RF 398 alpha t₂is isolated from E. coli transformants. This plasmid thus has the betahCG cDNA in a transcriptional unit under control of the mousemetallothionein promoter and the alpha hCG cDNA in a transcriptionalunit controlled by the SV40 early promoter.

Expression of Luteinizing Hormone (LH) cDNA Clones Construction of aHuman Pituitary cDNA Library

RNA is prepared from human pituitaries by homogenizing 5 to 10 grams ofthe frozen glands in 20 ml of a solution containing 4M guanidinethiocyanate, 1M 2-mercaptoethanol, 0.05M Na-acetate (pH 5.0), and 0.001MEDTA. One g CsCl is added per ml of homogenate and the suspension iscentrifuged at 2,000 rpm for 15 min. The supernatant is layeredcarefully over a 15 ml cushion of CsCl solution (containing 1.25 ml of1M Na-acetate (pH 5), 62.5 microliters of 0.4M EDTA and 39.8 g of CsClin a final volume of 35 ml) and centrifuged at 45,000 rpm in the Ti 70rotor of a Beckman ultracentrifuge for 18-24 h at 20° C. The RNA visibleas a pellicle in the gradient is removed with a syringe, diluted, andprecipitated by the addition of two volumes of ethanol. Following threecycles of dissolution and reprecipitation, the RNA pellet is dissolvedin H₂ O and brought to 0.01M Tris-HCl (pH 7.5) and 0.5M NaCl by theaddition of concentrated stock solutions. The preparation is thenenriched for poly A⁺ mRNA by two passes over oligo dT-cellulose, asdescribed above in the case of placental RNA.

A human pituitary cDNA library is constructed from the poly A⁺ mRNA asdescribed above for placental poly A⁺ mRNA except that both the largefragment E. coli DNA polymerase I and the avian myeloblastosis virusreverse transcriptase are used sequentially for second strand cDNAsynthesis. Reverse transcriptase is used first. The reaction is stoppedby phenol extraction. The aqueous phase of the centrifuged extract isapplied to a 5 ml column of BioGel A-5m. Fractions containing highmolecular weight material are pooled, concentrated, precipitated withtwo volumes of ethanol, dried, and dissolved in 100 mM Tris-HCl (pH8.3), 10 mM MgCl₂, 140 mM KCl, 20 mM 2-mercaptoethanol, 1 mM of each ofthe four deoxyribonucleoside triphosphates, for reverse transcription.Reverse transcriptase is added to about 20 units per microgram of cDNA.Double stranded cDNA is then treated with nuclease S1, tailed, andcloned as described above.

Isolation of Beta LH cDNA Clones

Colonies grown on nutrient agar plates containing 25 micrograms per mlof tetracycline are transferred to nitrocellulose filters. Colonies arelysed in situ by treatment with 0.5M NaOH and neutralized with 0.5MTris-HCl (pH 7.4) containing 1.5M NaCl. Liberated DNA is fixed to thefilter by baking at 80° C. in a vacuum oven for 2 h. The filters arescreened by hybridization to a ³² P labeled 88 base pair fragment of thebeta hCG clone corresponding to amino acids 16 to 45 of the mature hCGbeta chain, which has 29 of 30 amino acids in common with this region ofthe beta LH polypeptide (FIG. 6). Hybridization is carried out overnightat 32° C. in 50% formamide, 0.75M NaCl, 0.075M Na-citrate (pH 7.0), 2.5%dextran sulfate, 0.1% polyvinylpyrollidone, 0.1 mg per ml bovine serumalbumin, and at least 10⁵ cpm per filter of ³² P-labeled 88 bp beta hCGfragment. Filters are washed several times in 0.15M NaCl, 0.015MNa-citrate at 37° C. before autoradiography. One of the positiveisolated clones LH12 (FIG. 7), is used further. LH12 is 365 bp long andincludes sequences coding for 15 amino acids of the pre-beta signalsequence plus 105 amino acids of the mature beta LH polypeptide. Itsnucleotide sequence is determined. Since the complete mature beta LH isnot coded by LH12, further screening of the human pituitary cDNA libraryis carried out using a 240 bp NcoI-PvuII fragment of LH12 (FIG. 7) as a³² P labeled hybridization probe. The clone LH6 (FIG. 7) is isolatedfrom this screening. LH6 contains the complete 3' end of beta LH,including the region corresponding to the untranslated portion of themRNA through 27 A residues of the poly A "tail" of the mRNA. No clonesare found that extended further than LH12 in the 5' direction. DNAsequencing of the complete, combined mature beta LH coding regionsreveals two differences in the amino acid sequence of beta LH from thepublished protein sequence data: position 42 is a methionine andposition 55 is a valine. Also, the mature beta LH contains 121 aminoacids, based on the cDNA sequence.

A clone containing an intact signal peptide coding sequence and thecomplete mature beta LH sequence is constructed as shown in FIG. 8,using the restriction fragments illustrated in FIG. 7. A 104 bpEcoRI-DdeI fragment is isolated from the plasmid beta 579 H and ligatedto an isolated 181 bp DdeI fragment, subsequently digested with PstI,from the LH12 plasmid. Following ligation overnight at 15° C., theligation mix is digested with EcoRI and PstI and fractionated on a 7%polyacrylamide gel from which the desired 256 bp fragment is isolated.This fragment fuses the beta hCG signal sequence to that of the pre-betaLH in such a way as to provide a coding sequence for a 20 amino acidsignal peptide.

The 256 bp EcoRI-PstI fragment is cloned into pBR322 digested with EcoRIand PstI so as to give the plasmid LH beta 260. The 146 bp EcoRI-NcoIfragment indicated in FIG. 8 is isolated from a polyacrylamide gel andused later in the construction as described below.

The LH6 plasmid (FIG. 8) is digested with Sau3a and the 390 bp fragmentis isolated by polyacrylamide gel electrophoresis. This fragment is thendigested with HincII, ligated to BamHI linkers, digested with BamHI, andcloned into the plasmid pAPP at the BamHI site. pAPP is derived frompBR322 by digestion with AvaI, filling in the 5' overhang with thedNTP's and the large fragment DNA polymerase I of E. coli, digestionwith PvuII, and ligation to close the plasmid so as to eliminate thePvuII site. The plasmid LH6B, isolated from the ligation of the 340 bpBamHI fragment into the BamHI site of pAPP, is digested with EcoRI andPvuII, and treated with bacterial alkaline phosphatase. The fragmentsare ligated to a mixture of the 145 bp EcoRI-NcoI fragment of LH beta260, described above, and the isolated 241 bp NcoI-PvuII fragment fromthe plasmid LH12 shown in FIG. 8. The ligation mix is used to transformE. coli to ampicillin resistance. The plasmid LH 520 H/B is found amongthe transformants. LH 520 H/B contains a complete beta LH codingsequence including a hybrid signal peptide sequence.

Construction of p Alpha LHSVVP1

In order to express this pre-beta LH clone is an SV40-based vector, ashad been done for the pre-alpha and pre-beta hCG clones describedpreviously, it is desirable to place an EcoRI site very close to the ATGof the pre-beta coding sequence. This is accomplished by digesting LH520H/B with HgaI, filling in the 5' overhang, ligating on synthetic EcoRIlinkers, digesting with EcoRI and BamHI, and cloning the isolated 496 bpEcoRI-BamHI fragment into pBR322 digested with EcoRI and BamHI andtreated with bacterial alkaline phosphatase. The plasmid pLH496 R/B isisolated from E. coli transformed with this ligation mix and is used asthe source of the 496 bp fragment to be expressed.

The plasmid p alpha beta VPl, whose construction and use in expressionboth subunits of hCG is described earlier (FIG. 3), is digested withEcoRI and BamHI and ligated in a reaction mix containing the plasmidpLH496 R/B which had been digested with both of these enzymes (FIG. 9).The plasmid p alpha LHVPl is identified among the E. coli transformants.As shown in FIG. 9. the intact SV40 viral early region is taken from palpha 8VHVPl (FIG. 1) and inserted by ligation as a KsnI-SalI fragmentinto p alpha LHVPl which had been digested with KsnI and SalI to givethe plasmid p alpha LHSVVPl. By cutting this plasmid with BamHI andreligating, the virus alpha LHSVVPl is formed. This virus containscloned cDNA's for the common (to LH and hCG, as well as FSH and TSH)alpha subunit and the specific beta LH subunit under control of the SV40late promoter. The cloned cDNA's are positioned in such a way that thecommon alpha insert replaced the viral VP1 protein coding sequence andthe beta LH insert replaced the viral VP2 coding sequence.

Insertion of the Beta LH cDNA (With Beta hCG 5' End of Signal Peptide)into a BPV-Based Expression System

LH 520 H/B (FIG. 8) is digested with HindIII and BamHI, treated with theE. coli DNA polymerase (Klenow), ligated to synthetic SalI linkers,digested with SalI, and cloned into the SalI site of pBR322. Theresulting plasmid, LH 530 Sal, is used as a source of the LH cDNA clonefor insertion into the mouse metallothionein gene of the plasmid CL28 asdescribed in FIG. 10.

CL28 is cut with BslII, treated with nuclease S1, and ligated to XhoIlinkers. Following digestion with XhoI, ligation and digestion withBslII, E. coli is transformed with the reaction mix to give the plasmidCL28Xho. This plasmid is digested with BamHI and SalI and ligated to aBamHI plus 8alI digest of the plasmid pB2-2 (FIG. 4) to give the plasmidCL28XhoBPV. The latter LH insert is then ligated into the XhoI site ofCL28XhoBPV as a SalI fragment, since the 5' overhang of SalI digests iscomplementary to that of XhoI digests. Following digestion with XhoI toeliminate background, E. coli is transformed and the desired plasmidpCL28XhoLHBVP containing the (hybrid) pre-beta LH insert, in a BPV-basedplasmid, under control of the mouse metallothionein promoter, isisolated.

Transfection and Infection of Host Monkey Cells

The incorporation of virus-containing vectors into eukaryotic cells forthe production of a heteropolymeric protein is generally accomplished asfollows. First, if the viral DNA and homopolymeric protein-encoding DNAare incorporated into a plasmid, which is maintained, in, say, E. coli.the plasmid sequences (e.g. the pBR322 sequences) are removed and theresulting DNA is ligated to form circular DNA including the viral regionand the heteropolymeric protein-encoding sequence or sequences. Thiscircular DNA generally does not contain all of the genetic informationneeded to produce a replicating virus, the other necessary sequences(e.g. those encoding coat protein) having been replaced by theheteropolymeric protein-encoding sequence or sequences. The circularDNA, minus the plasmid DNA, must be close enough in size to thenaturally occurring viral DNA from which it is derived to permit the DNAto enter and replicate in appropriate host mammalian cells.

The circular DNA is used to transfect host cells in order to producevirus stock for later infections. Since some of the DNA necessary toproduce virus is missing, the transfection must occur in conjunctionwith helper virus DNA encoding enough of the missing function to producereplicating virus.

Transfected host cells are grown and incubated until lysed byreplicating virus. The resulting replicating virus stock, includinghelper virus, is then used to infect host cells for production of theheteropolymeric protein. Virus stock is maintained, since it generallyneeds to be reused to infect fresh batches of host cells, as eachculture of infected, protein-producing host cells generally iseventually lysed by the virus.

The specific recombinant DNA sequences described above are used totransfect, and then infect, host cells, as follows.

The pBR322 sequences are removed from the above-describedSV40-containing plasmids to produce transfecting viral DNA. In the caseof p alpha SVHVPl and p alpha beta SVVPI, this is accomplished bydigestion with BamHI, followed by ligation under conditions favoringcircularization of the fragments to give (among other products) alphaSVHVP1 and alpha beta SVVPI. For p beta 8VVPl, digestion with EcoRIfollowed by re-ligation brings the SV40 late promoter and VPl spliceregion into juxtaposition with the beta hCG cDNA insert at the same timethat it eliminates pBR322 sequences and forms beta SVPl. At the sametime, PtsA58 Bam (tsA58 SV40 viral DNA cloned into the pBR322 BamHIsite) is cut with BamHI and ligated to obtain self-ligated circles.Analogous methods are used for the LH vectors. Separate virus stocks areprepared as described below.

The DNA's, which are cut and ligated as described above, are ethanolprecipitated and dissolved in sterile water. Approximately 1 μg ofptsA58 Bam DNA (helper virus) and 10 μg of recombinant DNA (encodingalpha and/or beta hCG or LH) are combined in a sterile test tube, mixedwith 2 ml of TBS buffer (G. Kimura and R. Dulbecco 1972, Virology, 49,79-81) and 1 ml of 2 mg/ml DEAE-dextran solution and added to amonolayer of confluent monkey CV-1 cells previously washed twice with 10ml of TBS in a T-75 flask. The cells are left at 37° C. for 1-2 hrs withoccasional shaking, washed with TBS twice, fed with 10 ml of DMEMcontaining 5% fetal calf serum, and left at 40° C. for 10-15 days. Aftercomplete cell lysis, the medium is transferred to a test tube, frozenand thawed five times, and centrifuged at 3000 rpm for five minutes. Theresulting supernatants serve as virus stocks for infection of fresh SV-1cells.

To accomplish an infection, CV-1 cells are grown to confluence in aT-150 flask. 1 ml of one of the virus stocks (made as described above)is added to the flask and the cells are incubated at 40° C. for 5 days.

For mixed infections, CV-1 cells are grown to confluence in a T-150flask, alpha SVHVPl and beta SVVI viruses are mixed in a 1:1 ratio and 1ml of the mixed virus is used to infect CV-1 cells at 40° C.

Transfection of Mouse Cells

To produce heterodimeric hCG using a mixed transfection, five μg of eachBPV plasmid, i.e., pRF 375 (alpha hCG) and pRF 398 (beta hCG), are mixedand added to 0.5 ml of a 250 mM CaCl₂ solution containing 10 μg ofsalmon sperm DNA as carrier. This mixture is bubbled into 0.5 ml of 280mM NaCl, 50 mM Hepes and 1.5 mM sodium phosphate. The calcium phosphateprecipitate is allowed to form for 30-40 minutes at room temperature.

24 hours prior to transfection, 5×10⁵ cells of mouse C127 cells(available from Dr. Dean Hamer, National Cancer Institute, NIH,Bethesda, Md.) are placed in a 100 mm dish or T-75 flask. Immediatelybefore adding the exogenous DNA, the cells are fed with fresh medium(Dulbecco's Modified Medium, 10% fetal calf serum). One ml of calciumphosphate precipitate is added to each dish (10 ml), and the cells areincubated for 6-8 hours at 37° C.

The medium is aspirated and replaced with 5 ml of 20% glycerol inphosphate buffered saline, pH 7.0 (PBS) for 2 minutes at roomtemperature. The cells are washed with PBS, fed with 10 ml of medium,and incubated at 37° C. After 20-24 hours, the medium is changed andsubsequent refeeding of the cells is carried out every 3-4 days.Individual clones are grown in T-25 cm flasks. After 7-21 days, cellclones can be transferred to larger flasks for analysis.

To produce heterodimeric hCG using a single transfection, plasmid RF 398alpha t₂ is employed in the same manner as the above two plasmids wereemployed for a mixed infection.

To make heterodimeric LH, plasmids pRF 375 and pCL28XhoLHBPV are mixed,as described above in the case of hCG.

An interesting observation is that culturing cells containing beta hCGor beta LH-encoding vectors alone, in the absence of alpha-encodingcells, produces practically no beta subunit, while cells containingalpha and beta-encoding sequences produce not only heterodimer, but freebeta subunit as well. This lends support to the notion that theproduction of both subunits in a single cell culture has the additionaladvantage of somehow permitting the presence of the alpha subunit tostabilize the beta subunit.

A further interesting observation was that mouse C127 cells transformedby an expression vector for the alpha subunit of hCG, without theadditional vector for the beta subunit, produces an alpha subunit whichwill not associate with the complementary urinary beta subunit(combination<5%). The recombinant alpha subunit apparently has moreglycosylations than the native subunit and is thus prevented fromcombining with beta subunit to produce a biologically active hormone.However, when both subunits are produced by the same cells, properassociation and glycosylation occurs in vivo so that biologically activehormone substantially identical to the native hormone is expressed.

Isolation of the Human beta FSH Gene

A human genomic library in phage lambda (Lawn et al., Cell, 15,1157-1174 (1978)) is screened using "guessed" long probes. The ideabehind such probes, set forth in Jaye et al., Nucleic Acids Research,11, 2325 (1983), is that if the amino acid sequence of a desired proteinis at least partially known, a long probe can be constructed in whicheducated guesses are made as to the triplet encoding any amino acidwhich can be encoded by more than one, and not more than four, differenttriplets. Any correct guesses increase the amount of homology, andimprove the specificity, of the results.

To isolate desired regions of DNA, two labeled 45-mer probes are used:TB36, homologous with amino acids 56-70 of human beta FSH; and TB21,homologous with amino acids 73-87. These probes have the followingnucleotide compositions (corresponding amino acids are also given):##STR1##

The above probes are used to screen the human genomic library asfollows. TB21 is labeled with ³² P and used to screen approximately5×10⁵ lambda plaques on duplicate filters by the in situ plaquehybridization technique of Benton and Davis, Science, 196, 180-182(1977). The prehybridization solution is maintained at 55° C. forseveral hours and has the following composition: 0.75M NaCl; 0.15MTris/HCl, pH 8.0; 10 mM EDTA; 5×Denhardt's Solution; 0.1% sodiumpyrophosphate; 0.1% SDS; 100 microgram/ml E. coli t-RNA. Thehybridization solution has the same composition except that it ismaintained overnight at 45° C., and contains labeled probe in aconcentration of about 0.5×10⁶ cpm/ml. After hybridization, the filtersare washed four times in 1×SSC (=0.15M NaCl, 0.015M Na₃ -citrate) andexposed to x-ray film.

This screening procedure yields 50 plaques which hybridize to TB21 onboth sets of filters. These 50 individual plaques are picked andcombined into 10 culture pools containing 5 plaques each. The 10cultures are grown and DNA is isolated from 50 ml phage lysates. ThisDNA is then digested with EcoRI and fractionated on two identical 1%agarose gels. after which it is transferred to nitrocellulose paperaccording to the method of Southern, J. Mol. Biol., 98, 503-517 (1975).

The DNAs on the two filters are hybridized to ³² P labeled TB21 andTB36, respectively. Individual plaques from the pool containing arestriction fragment which strongly hybridizes to both probes are thenscreened according to the above procedure, except that the DNAs aredigested with EcoRI, BamHI, and EcoRI plus BamHI. In this way the 6.8 kbEcoRI-BamHI fragment containing human beta FSH is isolated.

A partial restriction map of clone 15B, containing the 6.8 kbEcoRI-BamHI fragment is shown in FIG. 2. In order to locate the positionof the beta FSH sequences within the clone, the 6.8 kb EcoRI-BamHIfragment of clone 15B is subcloned into pBR322 to yield plasmidp15B6.8R/B (FIG. 2). p15B6.8R/B is then digested with variousrestriction enzymes and the products are fractionated by agarose gelelectrophoresis using conventional methods. The DNA is blotted tonitrocellulose paper and hybridized to fragments of a porcine beta FSHcDNA clone labeled with ³² P by nick translation.

As shown in FIG. 2, the porcine beta FSH-probe hybridizes to only twofragments of the human DNA, namely a 1.1 kb HindIII-KpnI and a 1.4 kbPstI fragment. Partial DNA sequencing of these two fragments shows thatthis DNA indeed codes for human beta FSH and that the entire codingregion for beta FSH is contained in these two fragments.

As shown by the restriction map of FIG. 3, the beta FSH coding sequenceis interrupted by an intervening sequence of approximately 1.6 kbbetween amino acids 35 and 36 of mature beta FSH. The nucleotidesequence of the entire human beta FSH coding region and some of theflanking and intervening sequences are given below. The amino acidsequence deduced from the nucleotide sequence is given for the codingregion. ##STR2##

Still referring to the above sequence, there is a box around the ATGinitiation codon of the 18 amino acid signal peptide, which is cleavedpost-translationally. The mature protein begins with the amino acid Asnencoded by the circled triplet AAT. The exon-intron boundaries aremarked by arrows; they are flanked by the consensus sequence GT for thesplice donor and AG for the splice acceptor site. There is a box aroundthe stop codon TAA, the end of the coding region.

Below is a reproduction of the above sequence not broken into triplets,showing restriction sites; the ATG beginning and the TAA ending thecoding region are boxed and the exon-intron junctions are marked byarrows. ##STR3##

Insertion of the Beta FSH DNA into a BPV-Based Expression Vector

Referring to FIG. 3, a synthetic BamHI linker is inserted at the DdeIsite of p15B6.8R/B, which is located 42 nucleotides 5. of the ATGinitiation codon. Referring to FIG. 4, p15B6.8R/B is digested with DdeIand treated with E. coli DNA polymerase (Klenow), after which it isligated to synthetic BamHI linkers and digested with BamHI. The 295 bpfragment containing the first exon of FSH is isolated and cloned intothe BamHI site of pBR322. The resulting plasmid pBR295Bam is digestedwith KpnI plus EcoRI plus AvaI and ligated to p15B6.8R/B which has beendigested with KpnI plus EcoRI plus SmaI. The ligation mix is then usedto transform E. coli, and the plasmid pBR2.8Bam containing the humanbeta FSH DNA sequence as a BamHI fragment is identified from among thetransformants by restriction mapping.

As shown in FIG. 4, expression plasmid CL28FSH2.8BPV is preparedaccording to the same method used to prepare pRF375 (FIG. 1), exceptthat the 2.8 kb BamHI fragment of pBR2.8Bam is used in place of thealpha hCG cDNA clone. Plasmid CL28FSH2.8BPV can be used to transformmammalian host cells using conventional methods, and human beta FSH canbe isolated and purified.

Transfection of Mouse Cells

To produce heterodimeric FSH using a mixed transfection, five μg of eachBPV plasmid, i.e., pRF375 (alpha subunit) and CL28FSH2.8BPV (beta FSH).are mixed and added 0.5 ml of a 250 mM CaCl₂ solution containing 10 μgof salmon sperm DNA as carrier. This mixture is bubbled into 0.5 ml 280mM NaCl, 50 mM Hepes and 1.5 mM sodium phosphate. The calcium phosphateprecipitate is allowed to form for 30-40 minutes at room temperature.

24 hours prior to transfection, 5×10⁵ cells of mouse C127 cells(available from Dr. Dean Hamer, National Cancer Institute, NIH,Bethesda, Md.) are placed in a 100 mm dish or T-75 flask. Immediatelybefore adding the exogenous DNA, the cells are fed with fresh medium(Dulbecco's Modified Medium. 10% fetal calf serum). One ml of calciumphosphate precipitate.e is added to each dish (10 ml). and the cells areincubated for 6-8 hours at 37° C.

The medium is aspirated and replaced with 5 ml of 2 glycerol in phophatebuffered saline, pH 7.0 (PBS) for 2 minutes at room temperature.

The cells are washed with PBS, fed with 10 ml of medium, and incubatedat 37° C. After 20-24 hours, the medium is changed and subsequentrefeeding of the cells is carried out every 3-4 days. Individual clonesare grown in T-25 flasks. After 7-21 days, cell clones can betransferred to larger flasks for analysis.

Deposits

The following, described above, have been deposited in the American TypeCulture Collection, Rockville, Md.:

alpha beta SVVPl, ATCC VR 2077;

alpha SVHVPl, ATCC VR 2075;

beta SVVPl, ATCC VR 2075;

pRF 375 in C127 cells, ATCC CRL 8401;

pRF 398 in C127 cells, ATCC CRL 8401;

pCL28XhoLHBPV E. coli, ATCC 39475;

pRF 398 alpha t₂ in C127 cells, ATCC CL 8400.

The following, described above, has been deposited in the AgriculturalResearch Culture Collection (NRRL), Peoria, Ill. 61604:

CL28FSH2.8BPV in E. coli, NRRL B-5923.

Use

The transformed cell lines of the invention are used to produceglycosylated biologically active heterodimeric human fertility hormones.hCG and LH made according to the invention, for example, have a numberof well-known medical uses associated with human fertility. Furthermore,FSH can be used, alone or in conjunction with hCG or LH, to induceovulation or superovulation for in vitro fertilization. In addition,heterodimeric FSH, or the beta subunit alone, can be used in diagnostictests for fertility and pituitary functions.

Human fertility hormones produced by recombinant cells have theadvantage, compared to such hormones obtained from natural sources, ofbeing free from contamination by other human proteins, in particularother fertility hormones.

Other Embodiments

Other host cells, vectors, promoters, transforming sequences, andviruses can also be employed. The host cell employed generally isdependent on the vector being used. For example, when the vector is areplicating virus or a non-replicating viral DNA, the host cells arecells capable of being infected or transfected, respectively, by thosevectors; e.g., SV40-containing vectors require monkey host cells,preferably CV-1 cells. Where the cloning vector is a plasmid havingprocaryotic control sequences, prokaryotic host cells, e.g., E. coli,are used. Where the cloning vector is a plasmid having eukaryoticcontrol sequences, appropriate eukaryotic host cells, e.g., mouse C127cells, are used. Besides autonomously replicating vectors, suitablevectors can be used to insert the DNA into the genome of the host cell,as is well known to those skilled in the art. As is also well known inthe art, the vector may also contain an amplifiable marker, such asDHFR, particularly when used to transfect a DHFR⁻ CHO cell line.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology herein is for the purpose of description and not oflimitation.

We claim:
 1. A process for producing biologically active heterodimerichuman fertility hormone selected from the group consisting of humanchorionic gonadotropin, human luteinizing hormone and human folliclestimulating hormone, comprising:transforming a eukaryotic cell with atleast one vector constructed so as to insert into said cell DNA codingfor each of the two subunits of the hormone, the translation of the DNAof each said subunit being controlled by a separate promoter; andculturing said cell under conditions appropriate to permit expression ofthe hormone therefrom, wherein said eukaryotic cell is one which permitsappropriate post-translational modification of the subunits so producedsuch that the formed protein is biologically active.
 2. A process inaccordance with claim 1, wherein said hormone is human chorionicgonadotropin.
 3. A process in accordance with claim 1, wherein saidhormone is human luteinizing hormone.
 4. A process in accordance withclaim 1, wherein said hormone is human follicle stimulating hormone. 5.A process in accordance with claim 1, wherein said transforming stepcomprises transforming the cell with a single vector containing DNA forboth of said subunits under the control of separate promoters.
 6. Aprocess in accordance claim 1, wherein said transforming step comprisestransforming the cell with two vectors each containing DNA for one ofsaid subunits and an appropriate promoter.
 7. A process in accordancewith claim 1, wherein said at least one vector is autonomouslyreplicating.
 8. A process in accordance with claim 1, wherein said atleast one vector is a plasmid or a replicating virus.
 9. A process inaccordance with claim 1, wherein each of said promoters are different.10. A eukaryotic cell transformed by at least one vector constructed soas to insert into said cell heterologous DNA coding for each of the twosubunits of a heterodimeric human fertility hormone selected from thegroup consisting of human chorionic gonadotropin, human luteinizinghormone and human follicle stimulating hormone, the translation of theDNA of each said subunit being controlled by a separate promoter, saideukaryotic cell being of a species which permits appropriatepost-translational modification of the subunits so produced such thatthe formed protein is biologically active.
 11. A cell in accordance withclaim 10, wherein said hormone is human chorionic gonadotropin.
 12. Acell in accordance with claim 10, wherein said hormone is humanluteinizing hormone.
 13. A cell in accordance with claim 10, whereinsaid hormone is human follicle stimulating hormone.
 14. A cell inaccordance with claim 10, wherein the cell has been transformed with asingle vector containing DNA for both of said subunits under the controlof separate promoters.
 15. A cell in accordance with claim 10, whereinthe cell has been transformed with two vectors each containing DNA forone of said subunits and an appropriate promoter.
 16. A cell inaccordance with claim 10, wherein said at least one vector isautonomously replicating.
 17. A cell in accordance with claim 10,wherein said at least one vector is a plasmid or a replicating virus.18. A cell in accordance with claim 10, wherein each of said promotersare different.
 19. An expression vector containing DNA encoding each ofthe two subunits of a heterodimeric human fertility hormone selectedfrom the group consisting of human chorionic gonadotropin, humanluteinizing hormone and human follicle stimulating hormone, withseparate promoters for each said subunit.
 20. An expression vector inaccordance with claim 19, wherein said hormone is human chorionicgonadotropin.
 21. An expression vector in accordance with claim 19,wherein said hormone is human luteinizing hormone.
 22. An expressionvector in accordance with claim 19, wherein said hormone is humanfollicle stimulating hormone.
 23. An expression vector in accordancewith claim 19, wherein each said promoter is different.
 24. Anexpression vector in accordance with claim 23, wherein said twodifferent promoters are derived from two different species.
 25. Anexpression vector in accordance with claim 19, which is autonomouslyreplicating.
 26. An expression vector in accordance with claim 19comprising a plasmid or a replicating virus.